Method of killing B-cells in a complement independent and an ADCC independent manner using antibodies which specifically bind CDIM

ABSTRACT

Methods are provided for inducing cell death in B-cells, including neoplastic B-cells, by employing reagents that bind to a B-cell epitope. Particularly, antibodies specific for the marker can be administered to a host to induce death in B-cells to which the antibodies bind or can be used in ex vivo clinical situations to selectively remove B-cells. A B-cell specific oligosaccharide epitope useful as a B-cell marker has been identified. The ligand being recognized on B lymphocytes has no apparent similarities to any of the known pan-B cells markers. In addition, proteins which specifically bind the disclosed epitope are provided. Human monoclonal antibody 216, which recognizes this B-cell epitope, is cytotoxic to B-cells and binds all CD19 +   and CD20 +   B lymphocytes in human peripheral blood and spleen. Furthermore, MAb 216 does not distinguish B cells by the isotype expressed, binding IgG +  and IgM +   cells with equal intensity, and also bind all B cells regardless of their CD5 expression. Methods to inhibit neoplastic B-cell growth by administering a B-cell-cytotoxic protein are presented. These products and methods find use in diagnosis and therapy.

TECHNICAL FIELD

The field of this invention is the control of B-cell proliferation in amammalian host as a therapy.

BACKGROUND

The immune system is the first line of defense against many pathologies.Particularly, the lymphoid compartment is concerned with monitoringtumorigenesis, invasion by pathogens, such as bacteria and viruses,aiding in the removal of foreign bodies, and the like. Essential to theability of the lymphoid compartment to protect the host against thevarious pathologies is the ability to recognize self from non-self. Inmonitoring tumorigenesis, subtle distinctions may be involved and thehigh incidence of cancer, particularly in the aged, suggests that themonitoring frequently breaks down over time. In addition, because of theenormous diversity of the environment to which the immune system isexposed, there is always the possibility that epitopes will beencountered, which may trigger an immune response which can be directedagainst self. Other mechanisms may also be operative in the processwhere a lymphoid cell attacks an endogenous epitope. These autoimmunediseases can be extremely destructive, as is evidenced by diabetes,rheumatoid arthritis, neuronal diseases, such as multiple sclerosis, andthe like. While in many cases, the disease is associated with T-cellattack, in some of the diseases, there may be a B-cell component, and inother diseases, such as rheumatoid arthritis and lupus nephritis, theprimary mediator may be B-cells.

The lymphoid compartment may be more susceptible than other cells totumorigenesis, because of the recombinatorial processes associated withthe rearrangements involved with formation of immunoglobulins and theT-cell receptor. Lymphoid cancers, such as lymphomas and leukemias areparticularly dangerous, because of the opportunity for migration of thelymphoid cells throughout the body and the many sites in the periphery,where lymphocytes reside, so as to provide numerous opportunities formetastasis. Furthermore, these diseases interfere with the nativeprocess which is intended to monitor tumorigenesis.

There is, therefore, substantial interest in being able to developtechniques and therapies which will allow for selective reduction incell types associated with pathogenesis.

Relevant Literature

Grillot-Courvalin et at. (1992) Eur. J. Immunol. 22:178 1, describe ananti-B-cell autoantibody from Wiskott-Aldrich syndrome which recognizesi blood group specificity on normal human B-cells. The production ofhuman monoclonal antibodies is described by Bieber and Teng (1987), Invitro sensitization for the production of human monoclonal antibodies,in Human Hybridomas, A. J. Strelkauskas ed. Marcel Dekker, Inc., NewYork, p. 39. Kannagi et at. (1983) Cancer Res. 43:4997, describe factorsaffecting expression of glycolipid tumor antigens. Niemann et al. (1978)Biochem. Biophys. Res. Comm. 81:1286, describe Blood group i and Iactivities of "lacto-N-nor-hexaosylceramide" and its analogues,particularly the structural requirements for i-specificities.

SUMMARY OF THE INVENTION

Methods are provided for inducing cell death in B-cells, includingneoplastic B-cells, by employing reagents that bind to a 75 kDglycoprotein B-cell marker. Particularly, antibodies specific for themarker can be administered to a host to induce death in B-cells to whichthe antibodies bind or can be used in ex vivo clinical situations toselectively remove B-cells.

DESCRIPTION OF SPECIFIC EMBODIMENTS

In accordance with the subject invention, methods and compositions areprovided for killing B-cells, particularly neoplastic B-cells, incellular compositions comprising a plurality of cells, particularlyhematopoietic cells. The method allows for a therapy in the treatment ofthe aberrant proliferation of B-cells and for the selective removal ofB-cells from cultures or other ex vivo situations. By B-cells isintended those cells of the B-cell lineage, where B-cells may be definedas comprising surface membrane protein markers found on normal B-cells,such as CD19, CD20, CD21 and CD22.

The CDIM epitope is a three-dimensional structural conformationrecognized on normal human, peripheral, and splenic B-cells and on someneoplastic B-cells by the human monoclonal antibody 216. The epitope isdefined structurally in terms of spatial conformation, functionally interms of specific antibody binding, and cytologically in terms ofcellular distribution, as described below.

The spatial conformation of CDIM is characterized by being a hexose orlonger, straight-chain oligosaccharide comprising acyl-substitutedrepeating glucosamine subunits. The epitope is structurally related to,but distinct from, the "I" and "i" antigens present on adult and chordred blood cells (RBC's) respectively, and to antigens derived from lipidA, lipooligosaccharides of N. gonorrhoeae and N. meningitides, andchitin. Structurally related synthetic sugars include gentiobioseoctaacetate, trichitose, and lacto-N-norhexosyl ceramide.

The CDIM epitope can be identified on a B-cell surface usingfluorescent-labeled human monoclonal antibody (MAb), particularly thehuman monoclonal antibody 216. Cells carrying the epitope can beanalyzed, for example, by a fluorescence-activated cell sorter (FACS).For example, a human MAb can be biotin labeled and detected withfluorescent-labeled streptavidin, where control human MAbs do not bindto the human B-cells.

The CDIM epitope is naturally presented as the glyco-moiety of theglycoprotein known as gp75, an approximately 75 kD MW (as determined byconventional SDS-PAGE) glycoprotein found on substantially allperipheral B-lymphocytes and splenic B-lymphocytes and on certaincultured B-cell lymphoma lines, such as Lam, REH, and JY25. It is alsofound on 30-40% of primary B-cell lymphomas of various histopathologicclassifications. At least about 90% of these various categories ofcells, more usually about 100% of these cells, will present theindicated epitope. The antibody 216, which recognizes CDIM on B-cells asdescribed above, does not recognize this epitope on cultured T-cellssuch as Peer and HUT 78, macrophage lines such as U937, and some B-celllines such as TAB, Daudi, Ramos, and 8866, since the CDIM epitope is notpresent on normal T cells, macrophages, NK cells, epithelial,endothelial or mesenchymal cells.

For the purpose of this invention polyvalent (two or more binding sites)CDIM-binding receptors are required. By "receptor" is intended acompound which has a specific affinity for the CDIM epitope, generallyat least about 10⁻⁷ M, preferably at least about 10⁻⁸ M, so as to beable to bind to gp75. The polyvalent nature of the receptor allows thesimultaneous binding of at least two gp75 molecules on the cell membranesurface, thereby forming a cross-link. Conveniently, antibodies can beused from any of the immunoglobulin families, such as A, D, E, G, and M;it is not requisite that the antibody be associated with variouscytotoxic processes associated with particularly Fc-initiated processes.Usually, the antibody will be IgM, since the pentameric structure ofthis molecule allows cross-linking unhindered by steric interference.Binding of at least two gp75 molecules on the same cell surface by thesame receptor elicits a cellular response, resulting ultimately in celldeath. Besides antibodies, other receptors with the indicated affinitywill find use, where the receptor can, for example, be associated with alectin either naturally occurring or modified. Alternatively, smallsynthetic molecules can be devised which will allow for specific bindingand cross linking of the CDIM epitope. One may subject the variableregion of a MAb to mutagenesis to enhance the binding affinity of anantibody for the CDIM epitope, if desired.

The gp75- and CDIM-binding agents can be used in therapy for treatmentof B-cell proliferative diseases, such as B-cell neoplasia andautoimmune diseases. Thus, the subject agents will find application inthe treatment of autoimmune-mediated disease, particularlyB-cell-mediated disease. For example, the human MAb 216, by binding toat least two CDIM epitopes on the same cell, causes the death of thecell expressing this epitope. Epitope-mediated death does not requirecomplement or cell-mediated lysis. While, for the most part, human cellsin human patients will be a primary interest, other animals,particularly domestic animals, will also be served by the subjectmethodology to the extent that a given agent reacts across species,which is readily determined by binding studies of the type describedherein.

For therapeutic uses, the compositions and selective agents disclosedherein can be administered by any convenient technique, which can varydepending on the nature of the compound/agent, the purpose and frequencyof the treatment, and the like. For small molecular weight agents, oraladministration is preferred, and enteric coatings are indicated wherethe compound is not expected to retain activity after exposure to thestomach environment. Generally the amount administered will beempirically determined, typically in the range of about 0.1 to 1000 μgactive ingredient per kg of recipient, with adjustment by a physician orother person after consideration of clinical results.

Large proteins are preferably administered parenterally or systemically,conveniently in a physiologically acceptable carrier, e.g., phosphatebuffered saline, saline, or deionized water. Some agents such asantibodies can also be administered nasally. Typically, compositions areadministered to a retained physiological fluid such as blood. Otheradditives can be included, such as stabilizers, or bacteriocides. Theseadditives, if present, will be present in conventional amounts.

The subject agents can also be used for treating cell populations inculture to diminish the B-cell population, whether normal or neoplastic,in the culture. Thus, in mixed cultures, where one wishes to avoidinterference by B-cells, where one is interested in studyingantigen-presenting-cell mechanisms other than those associated withB-cells, where one is analyzing for cells associated with mediatingsecretion of a particular cytokine, or where one wishes to study a mixedcell population for other purposes without the presence of B-cells, thiscan be achieved by adding an amount of the subject agent effective toremove substantially all of the B-cells present in the culture. In asimilar manner ex vivo therapeutic treatments can be utilized in whichblood is removed from a patient into an external environment (as indialysis), treated to remove excess B-cells, and then returned to thepatient.

Where CDIM epitope-specific antibodies are administered therapeutically,it is desirable to minimize the likelihood of an immunogenic orallergenic response by using host-specific antibodies (e.g., humanantibodies in humans). While intact antibodies are commonly used, theantibodies may be modified in a variety of ways, by enzymatic cleavageto provide fragments, reduction of disulfide linkages, and the like.

In referring to an isolated component or compound, the isolatedcomponent or compound will constitute at least about 1%, usually atleast about 10%, and more usually at least about 50% by weight of theisolated material. By pure compound or composition is intended at leastabout 90%, usually at least 95%, and more usually at least about 99% byweight of the component or compound. Unless otherwise indicated,functional fragments will also be intended when referring to componentsor compounds.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES

Production, characterization, and conjugation of human MAbs.

Human MAb 216 was prepared by diffusion of uninvolved spleen lymphocytesfrom a patient with nodular lymphoma. The cells were incubated in vitrowith LPS and fused to the heteromyeloma line SHMD33. This antibody wasfound to be mu, lambda using peroxidase-labeled chain-specificantibodies (Cal Tag, South San Francisco, Calif.). Nucleotide analysisof the heavy chain showed it was encoded by the VH 4.21 gene.

Human MAbs were purified on high pressure liquid chromatography using acarboxymethyl column (BioRad, Richmond, Calif.). Hybridoma supernatantcontaining 1% FCS was diluted 1:4 with 20 mM Na acetate pH 5.5 The MAbswere eluted with 300 mM NaCl Tris buffer pH 8, dialyzed in PBS andconcentrated if necessary on a Centriprep concentrator (Areicon,Danvers, Mass.). By PAGE analysis the purified material was 85-90% IgMand also contained transferrin and BSA. Concentration of the purifiedimmunoglobulins was determined by sandwich ELISA using a humanpolyclonal IgM standard (Cooper Biomedical, Malvern, Pa.).

MAb 216 and other human IgM MAbs were biotinylated usingN-hydroxysuccinimidobiotin (Pierce, Rockford, Ill.) at a ratio of 60μg/mg IgM.

Flow cytometry

Human adult splenic mononuclear cells were obtained from patientsundergoing therapeutic splenectomy, and peripheral blood was obtainedfrom normal volunteers. Lymphoma cells were obtained by biopsy orlaparotomy for removal of tumor. All procedures had the approval of theCommittee for the Protection of Human Subjects at Stanford University.Spleens were gently teased apart in HBSS with 1% FCS and 0.2% DNase andpassed through sterile nylon membranes to obtain single-cellsuspensions. Peripheral blood and splenocytes were centrifuged at 800 gfor 30 min through a ficol/hypaque gradient (Histopaque-1077, Sigma, St.Louis, Mo.). The mononuclear cell population was washed three times inHBSS with 1% FCS, and resuspended in staining medium (RPMI with 3% FCS,1 mM EDTA, and 0.01M HEPES)at 2.5×10⁷ cells/mi.

Tumor tissue that had been removed from patients at surgery wasdisassociated into a cell suspension and frozen in DMSO with storage inliquid nitrogen. The cells were thawed and incubated overnight at 37degrees before staining. The thawed cells were also incubated 24 hourswith human MAb 216 or control human IgM MAbs and stained with propidiumiodide (PI) which measures cell death.

Multi-parameter flow cytometric analysis (FACS) has been described indetail (Parks et at. (1986) The Handbook of Experimental Immunology,supra, p. 29). Fluorescent-labeled mouse MAbs against CD epitopes werefrom Becton Dickinson. 5×10⁵ cells were suspended with predeterminedsaturating concentrations of each of the conjugated fluorescentantibodies in a final volume of 125 μl, and incubated on ice for 15 min.The cells were washed and resuspended in 200 μl of staining medium andanalyzed on a highly modified dual-laser FACS II (Becton Dickinson,Mountain View, Calif.), interfaced with a VAX 6300 computer (DigitalEquipment, Maynard, Mass.) running FAGS/desk software (Moore and Kautz(1986) The Handbook of Experimental Immunology. supra p. 30). Dead cellsare identified with the propidium iodide (1 μg/rnl) signal collected inthe APC- or TR-channel in experiments with three-colors (Parks et at.(1986) supra).

Endo-β-galactosidase treatment of cells

Peripheral blood B lymphocytes were incubated at 37° C. for one hour at5×10⁶ cells/ml in Iscove's with 5% FCS, with 15 mU/ml ofendo-β-galactosidase (Boehringer Mannheim, Indianapolis, Ind.). Thecells were washed and stained for FACS analysis. Cord RBCs at 50%concentration were incubated with 0.1 U/ml of endo-β-galactosidase at37° C. for 4 hours, washed and tested for hemagglutination.

RESULTS

216 MAb reacts with a carbohydrate ligand on human splenic andperipheral B lymphocytes.

Multiparameter FACS analysis of human mononuclear cells demonstratedthat the MAb binds specifically to all B lymphocytes (CD20⁺) obtainedfrom human spleen and adult peripheral blood. MS2B6, a human monoclonalIgM used as an isotype control, did not bind human B lymphocytes, nordid other poly-reactive natural antibodies.

The B lymphocytes reacting with 216 were also positive for other pan-Bcell markers, such as CD19, CD21, and CD22. Excess amount (10X) ofantibodies to CD19, CD20, CD21, CD22, and IgM did not inhibit thebinding of 216 to B cells. 216 does not distinguish between subsets of Blymphocytes, reacting with both CD5⁺ and CD5⁻ B cells. The MAb also didnot distinguish between the isotope expressed, reacting with bothsurface IgG or IgM beating B lymphocytes.

Mononuclear cells from human peripheral blood were treated withendo-β-galactosidase, and then stained with MAb 216. Reactivity to humanB lymphocytes is significantly reduced in enzyme-treated cells.Expression of an unrelated B cell marker (CD19) does not changefollowing enzyme treatment. Thus, sensitivity of both B lymphocytes andcord RBC to endo-β-galactosidase treatment, suggests that the epitoperecognized by the two antibody on B lymphocytes is also a carbohydrateantigen similar to the linear polylactosamine structure of the "i"antigen.

216 MAb binds to lymphoma cells

Twenty-seven primary lymphomas were analyzed by FACS. Twenty-three wereB-cell lymphomas. The MAb 216 did not react with any T-cell lymphomasand stained 10 of 23 B-cell lymphoma. The MAb 216 did not stain anysmall cleaved cell (follicular) lymphoma. The MAb 216 stained thefollowing classes of lymphoma; immunoblastic, diffuse large welldifferentiated, diffuse large cell, diffuse mixed, and diffuse smallcell.

In vitro B-cell toxicity of 216

Human MAb 216 is incubated in vitro at an Ab concentration of 10-20μl/ml with various types of cells. The cells are in tissue culture mediawith heat-inactivated normal human serum or in serum-free media and areincubated at 37° C. in 5% CO₂. After 18-24 hours incubation the cellsare stained with propidium iodide (PI) and/or Hoechst dye 33242 (andother Ab for determining type of cell) and analyzed on FACS. Cell deathis determined by uptake of PI and decreased DNA staining with Hoechstdye. When human spleen or peripheral blood lymphocytes are incubatedwith MAb 216, 60-80% of B cells are killed. Four primary lymphoma cellsuspensions that stained with the MAb 216, and two that did not, wereincubated 24 hours with either 20 μl/ml of the 216 MAb or control humanMAb in medial at 37 degrees in 5% CO₂. The cells were analyzed for celldeath using propidium iodide (PI) on FACS. The lymphoma cell suspensionsthat bound the MAb 216 showed significant PI uptake compared to thecontrol MAb. The two lymphoma cell suspensions that did not bind MAb 216did not take up PI. When B cell lymphoma lines are incubated with MAb216, 60-80% of the cells are killed. MAb 216 does not kill T cells, NKcells or monocytes. Other control human MAbs cause 0-5% cell death underthe same conditions.

Isolation and Characterization of gp75

Isolation

The B cell membrane proteins and glycoproteins including gp75 can beextracted and solubilized using a non-ionic detergent (such as TritonX). The B-cell membrane protein solution is passed over a column towhich the MAb 216 has been covalently bound using N-hydroxy succinimidecoupled to acrylamide beads (Affi Gel). The CDIM epitope is bound to the216 MAb and the gp75 is removed from the membrane solution. The columnis washed to remove other proteins. The gp75 can be released from thecolumn by competition with lacto-N-nor-hexosylceramide contained inliposomes which will competitive bind to the MAb 216.

Characterization of CDIM Ag

The MAb 216 binds to synthetic I/i antigen.

The following glycolipids were run on TLC: i antigen(lacto-N-norhexaosylceramide), sialyl i antigen, paragloboside(lacto-N-tetraocylceramide), sialyl paragloboside, I antigen (branched),and GM3. The MAb 216 was applied to the plate. The plate was washed andI¹²⁵ --labeled goat anti-human IgM was applied to the plate, washed andincubated with X ray film. The TLC plate was then sprayed with sulfuricacid and heated to visualize the glycolipids. Comparing the TLC plateand exposed film revealed that 216 bound i antigen, sialyl i antigen, Iantigen, and sialyl I antigen only. i Antigen(Lacto-N-nor-hexaosylceramide): Galβ 1-4 GIcNAcβ 1-3 Galβ 1-4 GIcNAcβ1-3 Galβ 1-4 Glcβ 1-Ceramide. Sialyl I Antigen: NeuAcα 2-3 Galβ 1-4GICNAcβ 1-3 Galβ 1-4 GLcNAcβ 1-3 Galβ 1-4 Glcβ 1-Ceramide. Paragloboside(Lacto-N-tetraocylceramide): Galβ 1-4 GIcNAcβ 1-3 Galβ 1- 4 Glcβ1-Ceramide. Sialyl Paragloboside: NeuAc α 2-3 Galβ 1-4 GIcNAcβ 1-3 Galβ1-4 Glcβ 1- Ceramide. GM3: NeuAc α 2-3 Galβ 1-4 Glcβ 1-Ceramide.

SUMMARY

216 binds all CD19⁺ and CD20⁺ B lymphocytes in human peripheral bloodand spleen. Furthermore, 216 does not distinguish B cells by the isotypeexpressed, binding IgG⁺ and IgM⁺ cells with equal intensity, and alsobind all B cells regardless of their CD5 expression. Accordingly, theligand being recognized on B lymphocytes is a novel glycoprotein, withno apparent similarities to any of the known pan-B cells markers.

It is evident from the above results and disclosure that a novel B-cellmarker and a specific oligosaccharide epitope thereof have beenidentified. In addition, novel proteins which specifically bind thedisclosed epitope are provided. These products and products derivabletherefrom find use in diagnosis and therapy. A deposit of the hybridomacell line secreting mAb 216 was made with the ATCC in Rockville, Md.USA, as Deposit No. HB 11659, on Jun. 14, 1994.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

What is claimed is:
 1. A method for killing B-cells within a mixedpopulation of cells, said method comprising:contacting said mixedpopulation of cells with an antibody/carrier compostion in which theactive ingredient consists of a cytotoxic amount of a monoclonalantibody that specifically binds CDIM, wherein the killing of B-cellsoccurs in a complement-independent and cell-mediated independent manner,and wherein the said mixed population of cells is outside a mammalianhost.
 2. The method of claim 1, wherein said monoclonal antibody is anIgM.
 3. The method of claim 1, wherein said B-cells are neoplastic. 4.The method of claim 1, wherein said mixed population of cells ishematopoietic.
 5. The method according to claim 1, wherein said B-cellsmediate an autoimmune disease.
 6. The method of claim 2, wherein themonoclonal antibody is the monoclonal antibody produced by the hybridomacell line ATCC HB
 11659. 7. The method of claim 1, wherein themonoclonal antibody is a human monoclonal antibody.